Optical Techniques in Biotechnology – Unit 1 Notes

Laser and Light in Biotechnology

  • Assumptions and course setup
    • Students should watch videos posted in GCR for basics and revision (Video-based learning and articles).

Laser and Ordinary Light

  • What is a laser?

    • Laser stands for Light Amplification by Stimulated Emission of Radiation.
    • It is typically mono-chromatic, directional, and coherent.
    • In contrast, ordinary light is generally multi-wavelength (polychromatic), non-directional, and incoherent.

  • Key contrasts (Laser light vs Ordinary light)

    • Mono-chromatic vs polychromatic.
    • Directional vs broad dispersion.
    • Coherent vs incoherent.

  • Visual cue (conceptual):

    • Laser light is shown as a narrow, focused beam with a single wavelength.
    • Ordinary light appears as many wavelengths with diffuse directions.

Basic Properties of Light and Interactions with Materials

  • Core interactions of light with materials (including tissue, bone, biological fluids):

    • Reflection
    • Refraction
    • Dispersion
    • Interference
    • Diffraction
    • Scattering
    • Polarization
    • Optical states: Opaque, Transparent, Translucent

  • Everyday example: Traffic scene

    • Incident light from cars is largely due to reflection off surfaces (road, vehicles, signs).
    • Refraction is present but secondary in this scenario.
    • Mirage formation on hot days is due to refraction caused by a hot, less dense air layer near the road surface changing the refractive index; light bends and creates water-like illusion.

  • Conceptual observation flow in tissue

    • Incident light → scattering → transmitted light (attenuated by reflection, absorption, and scattering) → reflected light
    • Example: Biological tissue shows both scattering and absorption affecting transmitted/reflected light.

Light Propagation in Biological Tissues

  • Light-tissue interaction is a complex interplay of:

    • Absorption: light energy converted to other forms by tissue constituents.
    • Scattering: redirection of light by tissue microstructures.
    • Refraction: bending of light at interfaces with different refractive indices.

  • Key tissue optical properties that quantify light-tissue interactions:

    • Absorption coefficient: \mu_a
    • Scattering coefficient: \mu_s
    • Refractive index: n (often called RI)

  • Purpose of these properties: to characterize how light propagates through living tissues for imaging and diagnostic techniques.

  • Reference context: Video 2 (GCR) discusses these optical properties in more detail.

Optical Properties: Normal Cells vs Cancer Cells

  • Optical property differences that are relevant for diagnostic/timely imaging:

    • Scattering Coefficient \mu_s
    • Normal cells: lower \mu_s; cancer cells: higher due to irregular structures, enlarged nuclei, dense organelles.
    • Absorption Coefficient \mu_a
    • Normal: typical absorption by hemoglobin, water, etc.
    • Cancer: often higher, particularly in the near-infrared (NIR) range.
    • Refractive Index (RI) n
    • Normal cells: RI ~ 1.36-1.38.
    • Cancer cells: RI ~ 1.38-1.42, elevated due to increased nucleic acid and protein concentration.

  • Overall implication: Cancer cells and normal cells show distinct optical signatures because of structural and compositional differences, affecting how light is absorbed, scattered, and refracted.

Wavelength-Dependent Tissue Penetration and Extinction

  • Tissue penetration depth depends on the wavelength of light.

    • Longer wavelengths generally penetrate deeper than shorter wavelengths.
    • This depth is governed by the extinction properties, i.e., absorption and scattering, which tend to be lower at certain NIR windows.

  • Representative wavelengths and depth interpretation (examples shown in figures):

    • 980 nm, 808 nm, 650 nm, 600 nm, 550 nm, 350–450 nm (range shown on graphs)
    • The tissue extinction and penetration depth are wavelength-dependent, with deeper penetration occurring at longer wavelengths in the near-infrared (NIR) range.

  • Practical takeaway: Longer wavelengths typically exhibit deeper penetration due to lower effective absorbance and scattering coefficients, i.e., lower extinction.

  • Visual interpretation prompts (checklist):

    • Tissue penetration is wavelength-dependent.
    • Longer wavelengths tend to penetrate deeper.
    • Depth dependence arises because longer wavelengths have lower absorption \mua and scattering \mus coefficients, resulting in lower extinction.
    • Light absorbance occurs when tissue absorbs energy during irradiation.
    • Light scattering reduces transmission intensity through tissue.

Wavelength Regions and Biological Windows

  • Electromagnetic spectrum regions relevant for biomedical optics:

    • UV, Visible (VIS), Near-Infrared (NIR) I and II bands.
    • NIR windows are advantageous for deeper tissue imaging due to reduced absorption by hemoglobin and water in certain ranges.

  • Depth and tissue type context (example bands):

    • Visible to NIR-I (roughly 650–950 nm) and NIR-II (roughly 1000–1700 nm) regions are used for deeper imaging.
    • Different tissue components (blood, water, fat) have distinct absorption characteristics across wavelengths.

  • Conceptual chart cues (from figures):

    • Depth increases with wavelength within certain windows, as absorption and scattering drop in those ranges.
    • Oxygenated vs deoxygenated blood show distinct absorption profiles across visible and NIR wavelengths.

Light-Tissue Interactions: Absorption, Scattering, and Refraction

  • Absorption in tissues

    • Due to chromophores such as:
    • Hemoglobin (oxy- and deoxy-): strong absorption in the visible range with peaks near approximately 420\,\text{nm}, 540\,\text{nm}, and 580\,\text{nm}.
    • Melanin: broad absorption spectrum that generally decreases with increasing wavelength.
    • Water: strong absorption in the infrared, with peaks around 1450\,\text{nm} and 1940\,\text{nm}.

  • Scattering in tissues

    • Caused by variations in refractive index among tissue components (cells, organelles, extracellular matrix).
    • Rayleigh scattering dominates when scatterers are much smaller than the wavelength of light (proteins, small organelles) and shows strong wavelength dependence.
    • Mie scattering dominates when scatterers are comparable in size to the wavelength (cells, nuclei) and shows weaker wavelength dependence with more forward scattering.

  • Refraction in tissue

    • Occurs at interfaces with different refractive indices, governed by Snell's law: n1 \sin\theta1 = n2 \sin\theta2
    • Multiple scattering events lead to a diffusion-like distribution of light within tissue.

  • Summary of interactions in tissue imaging

    • Light undergoes a combination of absorption, scattering, and refraction, shaping the detected signal used for imaging and diagnostics.

Chromophores and Their Spectral Signatures

  • Principal chromophores in the visible and near-infrared (NIR) ranges:
    • Hemoglobin (both oxy- and deoxy-): strong absorption with spectral features around 420 nm, 540–580 nm.
    • Melanin: broad, decreasing absorption with increasing wavelength.
    • Water: strong infrared absorption with peaks around 1450 nm and 1940 nm.
  • Practical implication: By selecting wavelengths that target specific chromophores, one can enhance contrast or selectively visualize particular tissue features.

Scattering Mechanisms and Light Propagation in Tissue

  • Rayleigh scattering
    • Occurs when scatterers are much smaller than the light wavelength.
    • Exhibits strong wavelength dependence (shorter wavelengths scatter more).
  • Mie scattering
    • Occurs when scatterers are comparable in size to the wavelength.
    • Exhibits weaker wavelength dependence and stronger forward scattering.
  • Implications for imaging
    • Scattering redistributes light within tissue, influencing resolution, contrast, and penetration depth.

Refraction and Light Diffusion in Tissue

  • Refraction
    • Light bends when crossing interfaces with different refractive indices, described by Snell's law: n1 \sin\theta1 = n2 \sin\theta2.
  • Diffusion and multiple scattering
    • Light undergoes many scattering events in tissue, leading to diffuse illumination and broadening of the light distribution.

Microscopy and Optical Transmission Through Materials

  • Properties of transparency, translucency, and opacity (from Read Before Next Activity)
    • Transmission:
    • Transparent objects: allow light to pass through with minimal scattering.
    • Translucent objects: allow partial light transmission with scattering.
    • Opaque objects: block light completely.
    • Light behavior:
    • Transmission: minimal scattering or absorption for transparent objects.
    • Scattering: significant scattering for translucent objects; helps enhance contrast.
    • Absorption or reflection: blocks light for opaque objects.
    • Microscopy imagery:
    • Transparent objects yield clear, high-contrast internal images.
    • Translucent objects yield blurred or diffused images with less detail.
    • Opaque objects show only surface or silhouette; no internal view.
  • Practical microscopy uses
    • Transparent samples are ideal for internal structure visualization (cell organelles).
    • Scattering-based contrast helps delineate contours and features by shadowing.
    • Opaque samples help block or shadow features to emphasize certain aspects.
  • Advantages and limitations
    • High resolution with minimal distortion for transparent specimens.
    • Scattering-based contrast aids feature outlining but can blur details.
    • Opaques prevent internal viewing, limiting information to surface features.

Practical Activities, Safety, and Ethical Considerations

  • Guided activities to observe light-tissue interactions
    • Activity: Observe leaves, flower petals, and water using basic light sources (mobile flashlight, LED, laser pointer).
    • Key prompts: transmission, absorption (glow/darken), reflection, refraction (through water), scattering/diffusion.
    • Safety: Do not shine lasers directly into eyes; handle tools carefully.
  • Activity 1 (peer learning): Virtual microscopy activity via OpenSTEM Africa app
    • Six different biological samples observed under varying magnifications; team members explain observations (6 minutes per team).
  • Safety and handling in experimental setups are emphasized throughout activities.

Transparent, Translucent, and Opaque Objects: A Reference

  • Definitions and behavioral guidelines (from Read Before Next Activity):
    • Transparent: allows complete light transmission; minimal scattering; sharp internal microscopy images.
    • Translucent: partial transmission with scattering; blurred internal details.
    • Opaque: blocks light; no internal view; surface or silhouette observable.
  • Practical microscopy uses and advantages:
    • Transparent: ideal for viewing internal structures like cell organelles.
    • Translucent: useful for enhancing contrast by scattering light.
    • Opaque: highlights contours or features through shadowing; useful for emphasizing surface characteristics.
  • Limitations:
    • Transparent samples may require staining to differentiate features.
    • Detail resolution is reduced in heavily scattering samples.
    • Opaque samples obscure what’s behind them.

Summary of Session Outcomes and Learning Resources

  • Session summary highlights:

    • Definition of light and its basic properties (as covered in videos uploaded to GCR).
    • Electromagnetic spectra overview (videos on GCR for prior preparation/post revision).
    • Light propagation and interaction with biological samples discussed in classroom learning.

  • Session Learning Outcomes (SLOs):
    1) State the basic properties of light.
    2) Interpret the electromagnetic spectra based on wavelength and energy.
    3) Demonstrate the interaction of light with biological samples.

  • References and further reading

    • The content includes references to specific literature and resources (e.g., Ref: 10.1002/adpr.202200098, Advanced Photonics Research).
    • Additional video resources: Video 2 (GCR) on tissue optical properties; videos on electromagnetic spectra; videos on light propagation in tissues.

  • Practical and ethical implications

    • Ethical use of light-based imaging in biotechnology relies on safe handling, appropriate targeting of wavelengths, and awareness of tissue damage risk (laser safety emphasized).
    • Real-world relevance includes biomedical imaging modalities (e.g., optical coherence imaging, near-infrared spectroscopy) that exploit tissue absorption and scattering properties for diagnostics and therapy planning.

  • End-of-unit quick references (key equations and terms)

    • Snell’s law: n1 \sin\theta1 = n2 \sin\theta2
    • Refractive index (RI): n\approx 1.36-1.38 for normal cells; n\approx 1.38-1.42 for cancer cells.
    • Absorption coefficient: \mua; Scattering coefficient: \mus; Optical depth and penetration are influenced by these values.
    • Chromophores and their spectral signatures: Hemoglobin (peaks near 420, 540, 580 nm), Melanin (broad decreasing with wavelength), Water (peaks near 1450 and 1940 nm).

  • Notable wavelengths and notes from the slides

    • Common wavelengths cited: 980\,\text{nm},\ 808\,\text{nm},\ 650\,\text{nm},\ 600\,\text{nm},\ 550\,\text{nm},\ 350-450\,\text{nm}
    • Wavelengths influence tissue depth penetration and imaging depth.

  • Links and resources mentioned in the transcript

    • Google Doc worksheet for the activity: https://docs.google.com/document/d/1beG0n39y1gAy9k3Kevkfw9h7XcULZWg1kBLuydRELds/edit?usp=sharing
    • Peer learning activity: OpenSTEM Africa virtual microscope app (Six samples): https://vlab.ug.edu.gh/OpenSTEMAfricamaterials/OpenSTEMAfricaapplications/virtual_microscope/index.html

  • Final reminder

    • Review the video content and familiarise with the electromagnetic spectrum, light-tissue interactions, and the optical properties of normal vs cancerous tissues to prepare for exam questions that require interpretation of figures and their implications for biomedical imaging.